Authors:

Antonio Barbáchano

Alberto Muñoz

Abstract

Cancer research, like many other medical investigations, strongly relies on model systems to decipher processes and mechanisms as well as for identification of targets and develop new and improved drugs and therapies. Classically, animal models and spontaneously or induced immortalized cell lines have been used in basic and translational oncology. These systems, however, do not adequately reproduce the in vivo situation, as inferred from the species-specific differences in gene expression, physiology, pharmacology, and microbiome, and the very low success of developed drugs in clinical trials. Organoids are three-dimensional cell cultures generated by multipotent adult stem cells or pluripotent (embryonic or induced in vitro) stem cells in the presence of an extracellular matrix that self-organize in tissue-like structures that partially reproduce features and function of the tissue of origin. Here, we describe the origin of organoid technology, in which European researchers were key players, the elucidation of their characteristics, and the development of the present and possible future applications of organoids. Organoids can be generated by distinct types of stem cells, normal or tumoral, derived from a variety of tissues and locations in the organism. Initially, organoids were of pure epithelial nature but a new generation of organoid cultures that either combine cell types including fibroblasts, endothelial or immune cells, or result from the fusion of distinct cell type organoids (assembloids) is in progress aiming to recapitulate in vitro the complexity of organs. Organoids are receiving huge interest as a model system for cancer and other pathologies, drug screening and regenerative and precision medicine.

Introduction

Organoids are three-dimensional (3D) structures derived from stem cells and their progeny growing in specific complex media and an extracellular matrix that can self-organize and differentiate into some cell types partially recapitulating features of the tissue of origin. Organoids can be generated by multipotent adult stem cells present in most tissues, pluripotent embryonic or induced stem cells, or by mutated stem cells present in tumours (cancer stem cells, CSC). They constitute an improved model system to study normal and pathological processes, and are being increasingly used in oncology for dissecting the tumorigenesis (genes, mutations and epigenetic alterations), biomarkers, antitumor drug discovery, biomarkers, action, resistance and toxicity, and the relation of tumours with the immune system and the microbiome. Organoids can also be established from organismal fluids, circulating tumour cells and tumour metastases.

Organoids are changing drastically biomedical research, paving the way in Oncology for personalized treatments of cancer patients. Available results after the first decade of organoid technology show that testing drug responses on tumour organoids (tumoroids) can help to identify treatment regimes. Attempts of precision medicine in oncology have up to now relied on the identification of actionable gene alterations. While only a very low proportion of cancer patients benefit from genomic-guided therapies (Marquart et al., 2018), drug activity testing in patient-derived tumour organoids constitute a direct alternative that does not require analysis of tumour mutational profile and provide valuable information of drug sensitivity. High concordance with patient response in the clinic has been reported (Bose et al., 2021; van Renterghem et al., 2023): organoids provide functional precision medicine. Importantly, patient-derived organoids directly generated from primary normal or cancer stem cells should be distinguished from forced aggregates of stem or non-stem cells cultured in suspension (spheroids), and, likewise, from those organoids obtained from mice grafted with human tumours. European contributions to organoid field research and its potential use in oncology and disease modelling have been abundant and significant, with various institutions and researchers responsible for pioneer discoveries and key advancements.

The History and Features of Organoids

Organoids were first established from intestinal tissue as a result of attempts to grow normal adult stem cells in culture in Hans Clevers' laboratory in Utrecht, The Netherlands. In 2009, following the identification of the leucine-rich, repeat-containing G protein-coupled receptor (Lgr5) as a marker of crypt bottom stem cells in the mouse small intestine and after the demonstration that these stem cells when acquire mutations are the origin of intestinal cancers, Clevers' group successfully isolated and cultivate stem cells generating as 3D structures that were called organoids (Sato et al., 2009). In 2011, the Clevers laboratory managed to generate human organoids. The success of these cultures was based on two key characteristics: the embedding of intestinal crypts or single stem cells in an extracellular matrix covered by a complex medium that together recreate the milieu (niche) existing at the colon crypt bottom, while minor modifications of the medium were used to generate organoids from cells disaggregated from colon adenomas and adenocarcinomas (CSC) (Jung et al., 2011; Sato et al., 2011). Within the extracellular matrix organoids form cystic structures that can be compact or contain an internal lumen to which the apical surface of intestinal stem cell-derived differentiated cells is oriented. Thus, organoids are composed by stem cells and their heterogeneous progeny of cells following differentiation programs, that finally die by apoptosis. The use of multipotent stem cells offers several advantages over the use of induced pluripotent stem cells: multipotent stem cells mimic the natural environment better since they have a more limited differentiation capacity and present greater genetic stability since they do not require reprogramming (lower risk of teratomas). Moreover, they can be more useful in precision medicine because recreate accurately the patient's tumour. Thus, multipotent stem cells are more suitable for modelling tumour behaviour and personalized treatment responses. Still, organoids derived from pluripotent cells are useful when organoids cannot be generated from adult stem cells, as is the case of the central nervous system.

Work done in the following years mostly at Clevers' laboratory and other laboratories in and outside Europe frequently run by scientists who were former Clevers' collaborators allowed to optimize culture conditions for colon organoids and to expand the repertoir of organoids derived from multipotent stem cells up to more than 20 normal and tumour tissues. Achievements in Clevers’ laboratory include the generation of human organoids derived from colon, prostate, liver, pancreas, breast, lung, urothelial, oral mucosal and cerebral organoids; and of mouse organoids from colon, stomach, pancreas, liver, prostate, urothelium, lacrimal gland and thymus organoids. Other European and non-European laboratories have described the generation of human organoids from colon, brain, fallopian tube, kidney, endometrium and cerebellum, and from liver and gastric tumours, as well as from from mouse organoids from salivary gland and urothelium (see Timeline section).

Organoids present many advantages as compared to cell lines and animal models. They allow the long-term growth of normal/non-tumour cells derived from healthy tissues as well as pre/post-treatment comparison and allow to reduce the number of experimental animals. Though the first generation of stem cell-derived organoids are pure epithelial and so, do not fully recapitulate the complexity of tissues/organs, they can capture functional features of human organs better than 2D cultures and animal models. Likewise, organoids generated from primary cancer stem cells retain the heterogeneity and features of original tumours better than immortal cancer cell lines, which are established with low success from very aggressive tumours, and an alternative to human tumours that are grafted in mice (patient-derived xenotransplants or PDX). PDX require more time, higher costs, and are less efficient than the establishment of organoids directly from patients. As discussed below, the new generation of organoids under development are heterotypic, including stromal (fibroblasts, vascular) and immune cells, and display technical advantages and culture conditions that substantially increase the physiological relevance of organoids.

Other advantages of organoids include: i) they are genetically stable during prolonged period of time, ii) the possibility of banking (to obtain so-called "Living Biobanks"), as they are long-term expandable and suitable for storage as can be subjected to cycles of freezing and thawing; iii) they allow longitudinal studies during the clinical management of cancer patients; and iv) organoids permit gene editing by using CRISPR technology or viral vectors to eliminate endogenous genes or introduce exogenous genes, either wild-type or mutated.

Organoids for Modelling Tumorigenesis and Metastasis

Organoids are useful tools to study the genetics basis of tumorigenesis. Models of human cancers can be generated by introducing crucial mutations (sequential or one step) in normal organoids. Due to its initial discovery, colon cancer organoids have been preferentially studied. The introduction in human colon normal organoids of mutated tumour suppressor genes or proto-oncogenes in a number of laboratories including those of Clevers and Sato confirmed the crucial role of several driver gene alterations (APC, TP53, SMAD4, RAS, PIK3CA) in colon cancer. Furthermore, using an elegant protocol involving the establishment of tumour organoids from mice carrying oncogenic driver gene mutations in their intestinal stem cells and the subsequent implantation in normal wild-type animals, Batlle's group demonstrated the importance of TGF- in the evasion of the immune system and the generation of colon cancer liver metastasis (Tauriello et al., 2018). Liver is the most common site of colorectal metastasis of this neoplasia and, intriguingly, contradictory roles for LGR5+ cells as responsible for this process have been proposed (Matano et al., 2015; Fumagalli et al., 2020). Again, the work in Batlle's laboratory has been crucial to assign to LGR5- cells the capacity to disseminate from primary colon tumors, whereas LGR5+ cells, probably resulting from dynamic phenotypic plasticity, are responsible for metastatic outgrowth. A distinct population of cells expressing epithelial membrane protein (EMP)-1 causes overt metastatic disease (Canellas-Socias et al., 2022). Remarkably, these authors also revealed that liver micrometastases are infiltrated with T lymphocytes that are excluded during the growth to macrometastases, which indicate the convenience of eliminating EMP-1+ cells and initiate T-cell-based immunotherapies at early steps of the process. Patients with colorectal cancer peritoneal metastases have limited therapeutic options and poor prognosis. Organoid drug testing to guide precision treatment in a personalized way for these patients was first used in a prospective multicenter study in Australia (Narasimhan et al., 2020), and later reproduced in Europe by Alberto Muñoz’ group (in Madrid, Spain) and Marcello Deraco and collaborators (in Milan, Italy).
Colon cancer patient-derived matched normal and tumor organoids have been used to investigate the proposed protective role of vitamin D in this neoplasia. Muñoz's laboratory has reported that the active vitamin D metabolite 1,25-dihydroxyvitamin D3 (calcitriol) variably reduce cell proliferation and induces partial differentiation in colon tumor organoids, whereas it inhibits proliferation more regularly and increase cell stemness in normal organoids (Fernandez-Barral et al., 2020). These results support a dual effect of calcitriol acting as an antitumor agent against colon cancer and as a homeostatic agent preventing the exhaustion of the stem cell population located at the colon crypt bottom that is responsible for the weekly renewal of intestinal epithelium during adult life. Brain organoids have been established from human tumors and also from embryonic or induced pluripotent stem cells from fibroblasts in which driver mutations are introduced, and from healthy human foetal brain tissue. Again, Clevers’ group and other European groups such as that of Madeline A. Lancaster are important players in this field (Lancaster, 2021; Hendriks et al., 2024). Current studies aim to solve the regional-specific cellular complexity and presence of non-neural cell types. While the importance of brain organoids to study brain patterning and function and to model neurological and neurodevelopmental disorders is evident, their application to cancer is focused to the investigation of the interaction of tumour cells and brain components and tumour invasion, drug testing and identification of therapeutic targets in glioblastoma and other brain neoplasias and diverse metastases (Wen et al., 2023).

##Organoids for Disease Modelling Beyond their widespread use in modelling tumorigenesis and metastasis, organoids have been employed to model infectious diseases, genetic and neurological disorders, and liver, intestine, kidney, heart, and bone marrow diseases. Organoids have proven to be a powerful platform for disease modelling, offering the possibility to study complex interactions in a controlled environment. Their use is expanding, promising advances in understanding disease mechanisms and developing new treatments.

Organoids for Drug Discovery, Biomarkers, Toxicity and Resistance

Immortal cancer cell lines have for long been used to identify cytotoxic compounds with low success rate, as most that were selected in vitro failed in early clinical studies. Once more, Clevers' group was pioneer showing that patient-derived organoids constitute a valid drug screening model system to test unlimited amounts of compounds and combinations (van de Wetering et al., 2015). Moreover, organoids have been used to investigate undesired effects of chemotherapeutic drugs such as 5-Fluorouracil or cis-platin and to identify biomarkers of resistance that can predict response and survival or cancer patients (Kong et al., 2020; Chen et al., 2022). Thus, Arne Van Hoek’s group (in Amsterdam, The Netherlands) described the induction by 5-Fluorouracil of secondary malignancies acting as their agents (Christensen et al., 2019). Furthermore, the parallel use of matched normal and tumor organoids from the same patient allows to investigate toxicity and therapeutic windows. Toxicity assays have also gained from the develop of 2D single layer patient-derived organoids that permit direct access to both the apical and basal surfaces of the epithelium and the co-culture of organoids with fibroblasts or immune cells.

##Organoids in Precision Oncology The initial collaborative study by Nicola Valeri and colleagues (in London, Great Britain) opened the analysis of the clinical impact of organoids on the personalized treatment of cancer patients (Vlachogiannis et al., 2018). These authors compared the response the therapy response of 23 colorectal and gastroesophageal heavily pretreated cancer patients and that of their tumor-derived organoids showing an 88% positive predictive value and a 100% negative predictive value. Later studies in a series of neoplasias have confirmed a good correlation between organoid response and cancer patient response to some, not all, chemotherapy drugs, demonstrating the utility of cancer patient-derived organoids as a tool for predicting response to therapies, with the advantage that the prior knowledge of the mutational profile of tumors is not required. Recent studies carried out in Europe include the collaborative study by G. Argilés and colleagues (in Naples, Italy, and Barcelona) in metastatic colon cancer (Martini et al., 2023), and those of Emile E. Voest and collaborators (in Amsterdam) (Ooft et al., 2021) and Sylvia F. Boj and colleagues (in Utrecht) (Smabers et al., 2024).

Organoids in Immunooncology Research

Immunotherapy is becoming a revolution in cancer treatment, offering durable responses and improved survival to a proportion of patients with certain malignancies. This unpredicted variability in the clinic makes personalized treatments an urgent demand. Organoids are finding an impact in the study of the antitumor action of the immune system. First steps in the implementation of organoids in precision immunooncology were performed by Emile E. Voest and colleagues, who reported the generation of tumor-reactive T cells in co-cultures of peripheral blood lymphocytes and tumor organoids and the initial testing of immune checkpoint inhibitors in organoids (Dijkstra et al., 2018). Today, a high number of studies are using organoids as a tool to study tumor immunosurveillance (interactions between immune and tumor cells) and testing immunotherapies, as recently reviewed by the groups of W.P.R. Verdurmen (in Nijmegen, The Netherlands) and Sarah J. Danson (in Sheffield, Great Britain) (Chernyavska et al., 2023; Rahman et al., 2024).

Regenerative medicine

Regenerative medicine aims to replace damaged tissues or organs to restore or maintain their normal functions. As a proof of concept, the groups of Clevers in the Netherlands and Watanabe in Japan demonstrated the feasibility of transplanting organoids grown in the laboratory to regenerate the intestinal epithelium of an animal model of inflammatory bowel disease. These pioneer studies showed that in vitro intestinal organoids could be transplanted into mouse damaged tissues to directly induce tissue regeneration. The development of organoid-based regenerative medicine is advancing rapidly, reaching the clinical trial phase.

Present Limitations of Organoids and Challenges

Despite the remarkable progress in organoid technology and the long list of their advantages with respect cell lines and experimental animals, a number of limitations and challenges remain unmet. The first is conceptual: organoids are said to be generated by stem cells, but it is today accepted that stemness is a transient, not fixed cellular state that may result from cell plasticity as a consequence of the interaction of cell genotype and epigenetic changes caused by external signals. Thus, perhaps the term "stem cell function" is more precise than "stem cells". In technical terms, a major limitation of first generation organoids is their pure epithelial nature, which clearly do not reproduce a tumor in vivo consisting in tumor cells and non-tumor or stromal cells that play important roles during tumorigenesis and modulate the response of tumor cells to therapies. Moreover, homotypic epithelial organoids do not allow to properly test anti-angiogenic or immune-based therapies. Drawbacks also include doubts about the capacity to reproduce tumor heterogeneity and their cystic architecture, that restricts size and lifespan as it requires passaging every few days, and it makes the apical cell surface non-accessible to drugs and other agents. Additionally, as discussed by Voest and collaborators, the implementation of organoid technology into clinical practice need to overcome several other limitations such as assay success rates and speed, reproducibility and standardization (van Renterghem et al., 2023).

New Generation Organoids

Several cell-coculture systems developing organoids that contain epithelial and stromal cells (fibroblasts, endothelial, immune) have been designed to better reproduce the in vivo tumor environment (improved matrix, heterotypic cell interactions, vascularization/angiogenesis...). Tissue engineering technologies based on organoids are creating sophisticated (microfluidics) devices that go further in modelling cancer ex vivo. In 2020, Matthias P. Lutolf and collaborators (in Lausanne, Switzerland) reported a microchip system consisting of a device with a central chamber covered with a complex scaffold permeable to gases, nutrients and macromolecules and flanked by reservoir chambers that supply medium and factors. This system, called organ-on-a-chip, allowed intestinal organoid culture mimicking the geometry of native crypts that enable the co-culture with immune cells, the unusual differentiation of rare Tuft and M-like cells and the colonization by microorganisms for modelling their interaction with host cells (Nikolaev et al., 2020). In a step further, Lutolf's group has recently developed a bioengineered human colon organoid system defined as "mini-colon" that enable drug testing and personalized medicine (Mitrofanova et al., 2024). This device allows the spatiotemporal controlled induction of colonic tumorigenesis ex vivo by integrating microfabrication, optogenetic and tissue engineering approaches. In this system, the initiation of colon tumors in a healthy environment can be tracked in real-time at the single-cell resolution by directing oncogenic activation through blue-light exposure in an apically open architecture that permit several weeks of continuous culture (Lorenzo-Martin et al., 2024). Assembloids are structures with higher-order of complexity. They are defined as self-organizing integrated cell systems arising by the combination of different types of organoids or of organoids with distinct specialized cell types. Thus, assembloids or assembled organoids constitute engineered-based, heterogeneous tissue-like structures enabling multicellular environments mimicking processes of tissue remodelling, tumor-immune interactions, tumor vascularization, and drug delivery (Mei et al., 2024). Bladder and lung cancer assembloids have been described aiming to reproduce cancer ecosystems such as brain metastasis of small-cell lung cancer (Qu et al., 2023). This new generation of organoid-based technologies are extremely interesting and promising for the study of cancer in the laboratory, but will require profound adaptations for their implementation in the clinic.

Conclusions

Doubtless, organoids will contribute as a valuable new tool in the armamentarium to fight against cancer. Organoid are applied to disease modelling and mechanism research and for drug screening, testing, and resistance in personalized medicine in an increasing number of cancer types (Harada et al., 2022; Ma et al., 2024). Organoid research in oncology represents a dynamic and evolving field, with European scientists playing a central role in its origin and shaping its trajectory. European researchers have made key/significant contributions to the organoid field, advancing our understanding of cancer biology and paving the way for the development of therapies tailored to individual patients.

Acknowledgements

We thank Drs. Elena Sancho and Eduard Batlle for their careful revision of the text.

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